Uplsapo10x

Confocal laser scanning microscopy (CLSM) observations were conducted with an upright FV1000-D confocal laser scanning microscope (Olympus). For fluorescein diacetate (FDA) and propidium iodide (PI) staining, samples were soaked in dye solution containing 5 μg/ml FDA, 10 μg/ml PI, and 100 mM sorbitol. After 1 min, the samples were observed via CLSM. For calcofluor white staining, samples were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 60 min under a vacuum at room temperature. The fixed tissues were washed twice for 1 min in PBS and cleared with ClearSee (Kurihara et al., 2015 (link)). The cleared samples were stained with 0.1% calcofluor white in ClearSee solution for 60 min and then washed with ClearSee solution for 30 min. The stained samples were observed via CLSM. UPLSAPO10X or UPLSAPO60XW (Olympus) was used as the objective lens. The excitation wavelength and transmission range for emission were 473 nm and 485 to 560 nm for FDA and green fluorescent protein (GFP), 559 nm and 617 to 717 nm for PI, and 405 nm and 425 to 475 nm for calcofluor white.

Images were derived from FV1000 confocal laser scanning microscope (Olympus) using UPLSAPO 10X NA:0.40, UPLSAPO 40 × 2 NA:0.95, UPLSAPO 60X O NA:1.35 objective lens and Axio zoom microscope (Carl Zeiss). Images were analyzed by ImageJ (NIH) program. Identical acquisition parameters were used for all the brain slices within a single set of experiment. Image analysis was not performed blind to the genotypes of animals. Inhibitory synapse was defined as co-localization between inhibitory presynaptic marker (VGAT) and inhibitory postsynaptic marker (Gephyrin). Puncta analyzer ver2.0 plugin for ImageJ was downloaded from github (https://github.com/physion/puncta-analyzer) and used to quantify the inhibitory synaptic puncta. The protocol using the Puncta analyzer is well described in the previous article^{62 (link)}.

Three microscope systems were used for the experiments in this study:i.

Olympus two-photon microscope system (FVMPE-RS) equipped with a 10× water-immersion objective lens (UMPLFLN10X; numerical aperture (NA), 0.30; working distance (WD), 3.5 mm) and a 25× water-immersion objective lens (XLPLN25XWML; NA, 1.05; WD, 2.0 mm). We used 920 nm and 1040 nm for the excitation of YFP and tdTomato, respectively.

ii.

Olympus confocal microscope system (FV1200MPE) equipped with a 10× air objective lens (UPLSAPO10X; NA, 0.4; WD, 3.1 mm), a 20× air objective lens (UCPLFLN20X; NA, 0.7; WD, 0.8 mm), and a 60× oil immersion objective lens (PLAPON60X; NA, 1.4; WD,0.12 mm). We used 405 nm, 488 nm, 594 nm, and 633 nm 1p lasers.

iii.

Zeiss confocal microscope system (LSM880) equipped with a 10× air objective lens (Plan-Apochromat 10X/0.45 M27), a 25× glycerol-immersion objective lens (LCI Plan-Neofluar 25x/0.8 Imm Korr DIC M27), a 20× air objective lens (Plan-pochromat 20x/0.8 M27), and a 63× oil immersion objective lens (Plan-Apochromat 63x/1.4 Oil DIC M27). We used 488 nm and 633 nm 1p lasers.